The ability to visualize biomolecules within living specimen by engineered fluorescence tags has become a major tool in modern biotechnology, cell biology, and life science. Encoding fusion proteins with comparatively large autofluorescent proteins is currently the most widely applied technique. As synthetic dyes typically offer better photophysical properties than autofluorescent proteins, alternative strategies have been developed based on genetically encoding unique tags such as Halo- and SNAP-tags, which offer high specificity but are still fairly large in size. Small tags like multi-histidine or multi-cysteine motifs may be used to recognize smaller fluorophores, but within the cellular environment they frequently suffer from specificity issues as their basic recognition element is built from native amino acids side chains. Such drawbacks may be overcome by utilizing bioorthogonal chemistries that rely on attaching unnatural moieties under mild physiological conditions.
Powerful chemistries that proceed efficiently under physiological temperatures and in highly functionalized biological environments are the copper(I) catalyzed Huisgen type (3+2) cycloaddition between linear azides and alkynes, the copper-free 3+2 cycloaddition between linear azides and strained cycloalkynes, or the inverse electron-demand Diels-Alder (4+2) cycloaddition reaction between a strained dienophile such as trans-cyclooctene or norbornene and a 1,2,4,5-tetrazine, both forms of click chemistry (Blackman et al., J. Am. Chem. Soc. 2008, 130, 13518-13519; Kolb et al., Angew Chem Int Ed Engl 2001, 40:2004; Devaraj et al., Angew Chem Int Ed Engl 2009, 48:7013; Devaraj et al., Bioconjugate Chem 2008, 19:2297; Devaraj et al., Angew Chem Int Ed Engl 2010, 49:2869; WO 2010/119389 A2; WO 2010/051530 A2). The standard (3+2) cycloaddition between an alkyne and an azide requires a copper catalyst that is toxic to bacteria and mammalian cells, which strongly reduces biocompatibility of this type of click chemistry. This limitation has been overcome by Bertozzi and co-workers, who showed that the click reaction readily proceeds without the need for a cell-toxic catalyst when utilizing ring-strained alkynes as a substrate (Agard et al., J Am Chem Soc 2004, 126:15046; WO 2006/050262 A2). Since then copper-free click chemistry has found increasing applications in labeling biomolecules. Fluorescent dyes comprising cyclooctynyl groups were used to label carbohydrates and proteins comprising enzymatically attached azide moieties in vivo (Chang et al., Proc Natl Acad Sci USA 2010, 107:1821) and the labeling of cycloalkyne-modified phosphatidic acid with azido fluorophores is described in Neef and Schultz, Angew Chem Int Ed Engl 2009, 48:1498. No catalyst was required in these applications.
Among the expanding repertoire of chemistries, in vivo chemistry applications of inverse Diels-Alder cycloadditions between tetrazines and strained dienophiles are attracting significant interest, particularly from those interested in performing live cell and animal imaging. Tetrazine ligations benefit from rapid, tunable kinetics as well as the existence of fluorogenic probes. Biomedical applications of tetrazine cycloadditions have been widely described and the implementation of tetrazine ligations to nanomaterial diagnostics has been addressed. For all this, see, for instance, the review of Seckute and Devaraj, Current Opinion in Chemical Biology 2013, 17, 761-767, and the references cited therein. Moreover, novel tetrazines and methods of synthesizing them are being developed (see, for instance, WO 2013/152359 A1). More specifically, WO 2011/095336 A2 describes methods and kits for the post-synthetic modification of nucleic acids by inverse Diels-Alder reaction, and WO 2013/029801 A1 describes methods for multiple orthogonal labeling of oligonucleotides by simultaneously performing an inverse Diels-Alder reaction and a copper-catalyzed click reaction. WO 2011/112970 A2 provides compositions and methods using bioorthogonal inverse electron demand Diels-Alder cycloaddition reactions for rapid and specific coupling of organic compounds to quantum dots (QDs).
The translational modification of proteins by direct genetic encoding of fluorescent unnatural amino acids using an orthogonal tRNA/aminoacyl tRNA synthetase pair offers exquisite specificity, freedom of placement within the target protein and, if any, a minimal structural change. This approach was first successfully applied by Summerer et al. (Proc Natl Acad Sci USA 2006, 103:9785), who evolved a leucyl tRNA/synthetase pair from Escherichia coli to genetically encode the UAA dansylalanine into Saccharomyces cerevisiae. In response to the amber stop codon TAG, dansylalanine was readily incorporated by the host translational machinery. This approach has meanwhile been used to genetically encode several small dyes and other moieties of interest. For instance, engineered Methanococcus jannaschii tyrosyl tRNAtyr/synthetase, E. coli leucyl tRNAleu/synthetase as well as Methanosarcina mazei and M. barkeri pyrrolysine tRNApyl/synthetase pairs have been used to genetically encode azide moieties in polypeptides (Chin et al., J Am Chem Soc 2002, 124:9026; Chin et al., Science 2003, 301:964; Nguyen et al, J Am Chem Soc 2009, 131:8720, Yanagisawa et al., Chem Biol 2008, 15:1187; WO 2013/108044 A2; WO 2002/085923 A2; WO 2002/086075 A2; EP2192185 A1).
The power of super-resolution microscopy (SRM) techniques heavily depends on the characteristics of the fluorophores. Most organic dyes have better photophysical properties and are typically more than 20 fold smaller than widely used fluorescent proteins. With recent advances in amber suppression technology, it is now possible to direct small, popular and commercially available fluorophores into specific protein residues. By means of an orthogonal tRNA/aminoacyl tRNA synthetase pair (tRNA/RS) from Methanosarcina mazei, unnatural amino acids (UAAs) carrying strained alkyne and alkene side chains are genetically incorporated at positions encoded by an amber (TAG) STOP codon (A. Borrmann, S. Milles, T. Plass, J. Dommerholt, J. M. Verkade, M. Wiessler, C. Schultz, J. C. van Hest, F. L. van Delft, E. A. Lemke, Chembiochem 2012, 13, 2094-2099; T. Plass, S. Milles, C. Koehler, C. Schultz, E. A. Lemke, Angew Chem Int Ed Engl 2011, 50, 3878-3881; T. Plass, S. Milles, C. Koehler, J. Szymanski, R. Mueller, M. Wiessler, C. Schultz, E. A. Lemke, Angew Chem Int Ed Engl 2012, 51, 4166-4170; K. Lang, L. Davis, S. Wallace, M. Mahesh, D. J. Cox, M. L. Blackman, J. M. Fox, J. W. Chin, Journal of the American Chemical Society 2012, 134, 10317-10320; S. Schneider, M. J. Gattner, M. Vrabel, V. Flugel, V. Lopez-Carrillo, S. Prill, T. Carell, Chembiochem 2013, 14, 2114-2118; WO 2012/104422). These modifications add only a few atoms to the amino acid side chain and can be placed freely within the protein, lowering the risk of functional impact. Subsequently, strained alkyne and alkene UAAs can undergo catalyst-free strain-promoted alkyne-azide cycloaddition (SPAAC) and [strain-promoted inverse electron-demand] 4+2 Diels-Alder cycloaddition (SPIEDAC) reactions with organic fluorophores carrying azide or tetrazine (Tet) functionalities, respectively. Both reactions are fully biocompatible. They are additionally orthogonal to each other, since azides only react with alkynes but not with alkenes (Y. Liang, J. L. Mackey, S. A. Lopez, F. Liu, K. N. Houk, Journal of the American Chemical Society 2012, 134, 17904-17907; M. R. Karver, R. Weissleder, S. A. Hilderbrand, Angew Chem Int Ed Engl 2011.).
While encoding a single UAA has become relatively straight-forward and incorporating more than one UAA has been described (US 2010/297693 A1; Han Xiao, et al., Angew Chem Int Ed Engl 2013, 52, 14080-14083) there is still a demand for robust and efficient multi-color labeling strategies in mammalian systems. At least two distinct strategies for UAA-based dual-color labeling and SRM are conceivable, which serve different experimental designs: i) Simultaneous incorporation of two different UAAs, harboring two orthogonal chemistries (e.g. SPAAC and SPIEDAC), recognizing each a different codon in a single protein (e.g. for Förster resonance energy transfer—FRET studies) or in two different proteins (e.g. for colocalization microscopy of two different molecules). ii) Sequential encoding of two different UAAs, harboring two orthogonal chemistries, in response to the same codon using a single tRNA/RS system. This can be done in a pulse-chase manner where the first UAA supplied to the growth medium is then chased by the second UAA. This can for example help to visualize protein sorting.
Despite large efforts, there is still a high demand for strategies to facilitate site-specific labeling of proteins in vitro and in vivo and robust multi-color labeling strategies in mammalian systems in particular. For practical reasons, it would be helpful if bioorthogonal coupling reactions proceeded with extremely rapid kinetics (k>102 M−1 s−1) and high specificity. Improving kinetics would minimize both the time and amount of labeling agent required to maintain high coupling yields. Thus, it was an object of the present invention to design extremely rapid bioorthogonal coupling reactions between tetrazines and dienophiles which allow establishing multi-labeling strategies. More specifically, it was an object of the present invention to provide amino acids or analogues thereof that can be translationally incorporated in polypeptide chains and allow labeling of the resulting polypeptide in vitro and in vivo as well as establishing multi-labeling strategies.